Phosphinic pseudo-peptide derivatives for the selective inhibition of the active c-terminal site of angiotensin converting enzyme (I) (ace)

ABSTRACT

The invention relates to the use of phosphinic pseudopeptide derivatives for production of a medicament for selectively inhibiting the active C-terminal site of angiotensin I converting enzyme. These derivatives are of formula (II):  
                 
 
where, R 1  may be a protecting group for amino functions usually used in peptide chemistry or an amino acid or a peptide protected by the above type of protecting groups, R 2  and R 3  correspond to a natural or unnatural amino acid side chain and R 4  and R 5  represent a hydrogen atom or a counterion. 
Applications: prevention and treatment of cardiovascular diseases in humans.

TECHNICAL FIELD

The present invention relates to the use of phosphinic pseudopeptidederivatives for the preparation of a medicinal product for selectivelyinhibiting the C-terminal active site of human angiotensin I convertingenzyme (ACE), i.e. without affecting the N-terminal active site of ACE.

Such a medicinal product may be used in the prevention and treatment ofvarious cardiovascular pathologies in man.

The present invention also relates to novel phosphinic pseudopeptidederivatives, to pharmaceutical compositions containing them and toprocesses for preparing the said phosphinic pseudopeptide derivatives.

PRIOR ART

Angiotensin I converting enzyme (ACE) is a central component in theregulation of arterial pressure and in the homeostasis of the variousphysiological functions of cardiovascular tissue. These actions appearto depend partly on:

-   -   the maturation of a powerful vasoconstrictor, angiotensin II,        via cleavage of the C-terminal end of angiotensin I, the        inactive peptide, with ACE, and    -   the degradation via ACE of a powerful vasodilator, bradykinin.

These actions are illustrated below.

Arterial hypertension, and also cardiac tissue diseases, result fromderegulation of the various vasoconstrictive hormones. Re-establishingthe equilibrium between vasoconstrictors and vasodilators, for thebenefit of the latter, is one of the main therapeutic objectives of themedicinal products used in human clinical treatment to remedy arterialhypertension and cardiac tissue diseases. It is thus understood how theinhibition of ACE can participate towards these objectives, bypreventing the formation of angiotensin II and potentiating bradykinin.ACE inhibitors are used in human clinical treatment not only to reducearterial hypertension, but also to preserve the functions of cardiactissue, as described in references [1] and [2].

The cloning of ACE followed by the determination of its primarystructure showed, surprisingly, the presence of two active sites in thisenzyme, as described in reference [3]. Via site-directed mutagenesis, ithas been possible to prove that these two active sites are fullyfunctional, i.e. capable of cleaving physiological substrates of ACE,such as angiotensin I and bradykinin (references [4] and [5]).

Despite all the studies performed for more than 20 years on ACE, it isstill not known whether the presence of two active sites in mammalianACE, resulting from duplication of an ancestral gene, corresponds to aparticular functional role. However, the recent discovery that, in vivo,in man, the peptide Ac-SDKP (N-acetyl Ser-Asp-Lys-Pro) is essentiallycleaved via the N-terminal active site of ACE, argues in favour of adistinct functional role for each of the active sites of ACE (reference[6]).

These considerations have led researchers to attempt to developinhibitors capable of highly selectively discriminating between the twoactive sites of ACE, in order to furnish tools capable of establishingin vivo the functional role of each of the sites of ACE.

In this regard, it is important to underline that all the inhibitorsused to date in clinical studies are mixed ACE inhibitors, i.e.inhibitors that simultaneously block the two active sites of ACE.

The first inhibitor that selectively blocks the N-terminal site of ACE,RXP407, which is a phosphinic pseudopeptide, has recently been developed(references [7] and [8]). This inhibitor, which is not metabolized inrats and mice, is, moreover, capable of inhibiting the degradation ofthe peptide N-acetyl Ser-Asp-Lys-Pro (Ac-SDKP) in vivo in mice(reference [9]). Thus, the injection of RXP407, by blocking theN-terminal site of ACE, would prevent the in vivo degradation ofAc-SDKP.

This inhibitor forms the subject of a preclinical study in the saidanimal, which is directed towards demonstrating its usefulness forprotecting haematopoietic tissue during chemotherapy treatment.

However, no inhibitor capable of discriminating between the two activesites of ACE by interacting essentially with the C-terminal site of ACEhas been described to date. However, it would be desirable to haveavailable such inhibitors. The reason for this is that, besides theirvalue in experimental and clinical research, they would have the majoradvantage, over standard mixed ACE inhibitors, of not interfering withthe physiological functions associated with the activity of theN-terminal site of ACE, for instance the metabolism of the peptideAc-SDKP.

It has been demonstrated that phosphinic peptides represent a genericfamily of compounds capable of very strongly inhibiting zincmetallopeptidases, the peptidase family to which ACE belongs, as may beseen in references [9] to [16]. In these compounds, the role of the PO₂⁻ group is to interact with the zinc atom located in the active site ofthese enzymes.

Besides the presence of the PO₂ ⁻ group, the chemical nature of theresidues P2, P1, P1′ and P2′ plays a deciding role in ensuring theselectivity of the interactions between a particular phosphinic peptideand a given zinc metallopeptidase (see references [8], [12] and [13]).Thus, the presence of quite specific residues in positions P2, P1, P1′and P2′ makes it possible to obtain selective inhibitors, which inhibitonly certain zinc metallopeptidases. Such selectivity may be anessential factor in the context of an in vivo use of these inhibitors.Specifically, it is estimated that the in vivo toxicity of certaininhibitors is predominantly due to their lack of selectivity for a giventarget.

According to these principles, the search for inhibitors capable ofselectively blocking the C-terminal site of ACE consisted inidentifying, in the family of phosphinic compounds, particular residueslocated in positions P1, P1′ and P2′, which give the inhibitors thecapacity to selectively interact with the C-terminal site of ACE.

DESCRIPTION OF THE INVENTION

This research has led to the discovery that the presence of apseudoproline residue in phosphinic pseudopeptides constitutes anessential element for obtaining selective inhibitors of the C-terminalsite of ACE.

Thus, one subject of the present invention is the use of at least onephosphinic pseudopeptide derivative comprising the amino acid sequenceof formula (I) below:

in which:

-   -   R₂ and R₃, which are identical or different, represent the side        chain of a natural or unnatural amino acid, the sequence:        also possibly forming the Pro (proline) residue, and    -   R₅ represents a hydrogen atom, a pharmacologically acceptable        counterion, or a group capable of forming an in vivo        hydrolysable phosphinic ester;        for the manufacture of a medicinal product capable of        selectively inhibiting the C-terminal site of angiotensin I        converting enzyme.

In this sequence, the PO₂ group may be in the form PO₂ ⁻ by beingassociated with a hydrogen atom or a pharmacologically acceptablecounterion, for example K⁺, Na⁺, NH₄ ⁺ or any other pharmacologicallyacceptable metal or non-metal ion. The nature of the counterion isirrelevant since, in water, the charged groups are dissociated.

The PO₂ group may also be in the form of phosphinic ester hydrolysablein vivo. In this case, the pseudopeptide is of the prodrug type, and,after in vivo hydrolysis of the ester, it generates the active form ofthe pseudopeptide.

Groups of this type that may be used for R₅ are described in particularin reference [20].

Examples of such groups that may be mentioned are groups correspondingto the following formulae:

In these formulae, t-Bu represents the tert-butyl group.

According to one particular embodiment of the invention, the phosphinicpseudopeptide derivative corresponds to formula (II) below:

in which:

-   -   R₁ represents a protecting group for an amine function, or an        amino acid or a peptide protected with a protecting group for an        amine function,    -   R₂ and R₃, which may be identical or different, represent the        side chain of a natural or unnatural amino acid, the sequence:        also possibly forming the Pro residue,    -   R₄ represents a hydrogen atom or a pharmacologically acceptable        counterion, and    -   R₅ is as defined above.

In the formulae given above, R₂ and R₃ represent the side chain of anatural or unnatural amino acid, for example a pseudoamino acid.

The natural amino acids may be chosen from alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, norleucine, lysine, methionine,phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan,tyrosine, valine, nitrophenylalanine, homoarginine, thiazolidine anddehydroproline.

A pseudoamino acid may be defined as an amino acid in which the amino orcarbonyl function has been replaced with another chemical group.

In formula (II) mentioned above, the group R₁ may be a common protectinggroup for an amine function in peptide chemistry. Such protecting groupsare well known to those skilled in the art and are illustrated, forexample, in the book entitled “Protective Groups in Organic Synthesis”,Second Edition, T. W. Greene and P. G. M. Wuts, John Wiley & Sons, Inc.,pages 309-315 [17]. As examples of such groups that may be used in theinvention, mention may be made of acetyl and benzyloxycarbonyl groups.

R₁ may also represent a natural or unnatural amino acid or a peptidewhose terminal amine function is protected with a common protectinggroup such as those described above.

According to the invention, as will be seen herein-below, the presenceof the pseudoproline residue is essential to obtain the selectivitytowards the C-terminal active site of ACE, but the nature of the sidechains present in R₂ and R₃ also plays an important role in theselectivity of the interactions of the derivatives used according to theinvention with the N-terminal and C-terminal sites of ACE.

Good results as regards the inhibition of the C-terminal site of ACEhave been obtained with pseudopeptides in which the group R₂ representsthe benzyl, methyl or phenylethyl group, i.e. the side chain ofphenylalanine, alanine and homophenylalanine.

For R₃, good results have been obtained when R₃ represents the sidechain of alanine, arginine or tryptophan, or when the sequence—NH—CH(R₃)—CO— represents a Pro residue.

Generally, R₄ and R₅ represent a hydrogen atom.

According to one preferred embodiment of the invention, the phosphinicpseudopeptide derivative corresponds to the following formula:

The phosphinic pseudopeptide derivatives that may be used in accordancewith the invention have been found to be capable of selectivelyinhibiting the C-terminal active site of angiotensin I convertingenzyme, and thus of controlling in vivo the physiological levels ofangiotensin II—which plays a central role in controlling arterialpressure and homeostasis of the cardiovascular functions in man—without,however, interfering with the metabolism of bradykinin or with that ofthe peptide Ac-SDKP.

Their use as active principles in a medicinal product is thus liable tofind numerous applications in the prevention and treatment of humancardiovascular pathologies, and especially pathologies in whichbradykinin is thought to take little part, for instance the preventionof atherosclerosis.

Among the phosphinic pseudopeptide derivatives whose use is envisagedaccording to the invention, some of them have never been described inthe literature.

Thus, a subject of the invention is also a phosphinic pseudopeptidederivative comprising the amino acid sequence of formula (I) mentionedabove, in which:

-   -   R₂ represents the side chain of a natural or unnatural amino        acid,    -   the sequence:        forms the Pro residue:        and    -   R₅ represents a hydrogen atom, a pharmacologically acceptable        counterion or a group capable of forming an in vivo hydrolysable        phosphinic ester.

Among these derivatives, the ones that are especially preferred arethose corresponding to the formula (II) mentioned above, in which:

-   -   R₁ represents a protecting group for an amine function, or an        amino acid or a peptide protected with a protecting group for an        amine function,    -   R₂ represents the side chain of a natural or unnatural amino        acid,    -   the sequence:        forms the Pro residue:    -   R₄ represents a hydrogen atom or a pharmacologically acceptable        counterion,    -   R₅ represents a hydrogen atom, a pharmacologically acceptable        counterion or a group capable of forming an in vivo hydrolysable        phosphinic ester.

More particularly, the preferred phosphinic pseudopeptide derivative isthe one of formula:

A subject of the invention is also a pharmaceutical compositioncomprising at least one phosphinic pseudopeptide derivativecorresponding to formula (II) mentioned above, in which:

-   -   R₂ represents the side chain of a natural or unnatural amino        acid,    -   the sequence:        forms the Pro residue:        and    -   R₅ represents a hydrogen atom, a pharmacologically acceptable        counterion or a group capable of forming an in vivo hydrolysable        phosphinic ester.

In this composition, the phosphinic pseudopeptide derivative preferablycorresponds to the formula:

With regard to the text hereinabove, such a pharmaceutical compositionis especially liable to find numerous applications in the prevention andtreatment of various cardiovascular pathologies in man.

The phosphinic pseudopeptide derivatives corresponding to formula (II)mentioned above, in which R₄ and R₅ represent a hydrogen atom, may beprepared via a process comprising the following steps:

-   -   1) reacting a compound of formula (III):        in which R₁ and R₂ are as defined above, with the compound of        formula (IV):        in which Ac represents the acetyl group and Et represents the        ethyl group, to obtain the compound of formula (V):    -   2) converting compound (V) into compound (VI) by reacting        compound (V) with sodium borohydride:    -   3) protecting the hydroxyl group of compound (VI) with an        adamantyl group Ad to give the compound of formula (VII):    -   4) saponifying compound (VII) to give the compound of formula        (VIII):    -   5) coupling the compound of formula (VIII) with the amino acid        of formula (IX) or (X):        in which R₃ is as defined above, and    -   6) removing the protecting group Ad.

According to this process, the phosphinic block of formula (VIII)comprising the pseudoproline is first synthesized, and peptide couplingof this phosphinic block with the desired amino acid is then performed.

Advantageously, the peptide coupling step 5) is performed by solid-phasepeptide synthesis using as solid phase a resin substituted with theamino acid of formula (IX) or (X), the N-terminal end of which will havebeen protected beforehand with an Fmoc (fluorenylmethoxycarbonyl) group.

If necessary, the phosphinic function of the pseudopeptide of formula(II) in which R₅ represents a hydrogen atom may then be esterified orsalified, by reacting it with suitable reagents.

The esterification may be obtained by coupling with an alcohol offormula R₅OH in which R₅ represents a group capable of forming an invivo hydrolysable phosphinic ester, for example using the processdescribed in reference [20] (method A).

The esterification may also be performed by reaction with a halide offormula R₅X in which R₅ represents a group capable of forming an in vivohydrolysable phosphinic ester. This reaction may be performed underalkaline conditions using the process described in reference [20](method B).

Before performing this esterification, the carboxylic acid function(s)of the pseudopeptide is (are) protected with suitable protecting groups,which are subsequently removed using standard techniques.

When it is desired to salify the phosphinic function of thepseudopeptide of formula (II) in which R₅ is a hydrogen atom, to replacethis hydrogen atom with a pharmaceutically acceptable counterion, thepseudopeptide is reacted with a suitable base containing thiscounterion, for example NaOH, KOH or NH₄OH.

The same technique may be used to salify the terminal carboxylic groupof the pseudopeptide, in order to replace the hydrogen atom with apharmaceutically acceptable counterion.

Other characteristics of the invention will emerge more clearly onreading the rest of the description that follows, which relates toexamples for preparing pseudopeptide derivatives in accordance with theinvention and for demonstrating their properties, with reference to theattached drawings.

Needless to say, this description hereinbelow is given for illustrativepurposes and with no limitation of the subject of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of synthons that are useful for thepreparation of the phosphinic pseudopeptides in accordance with theinvention.

FIG. 2 shows the chromatogram obtained during the purification of thepseudopeptide G by high performance liquid chromatography (HPLC).

FIG. 3 shows the in vivo effects of pseudopeptide G on the cleavage ofangiotensin I to angiotensin II.

FIG. 4 shows the in vivo effects of pseudopeptide G on the cleavage ofbradykinin.

FIG. 5 shows the in vivo effects of pseudopeptide G and of RPX407(selective inhibitor of the N-terminal site of ACE) on the cleavage ofthe peptide Ac-SDKP.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The synthesis of the phosphinic pseudopeptides was performed accordingto the synthetic scheme described in FIG. 1.

This figure shows the steps of the process resulting in the synthons ofthe formula (VIII):

in which:

-   -   R₁ represents the benzyloxycarbonyl (Cbz) group, and    -   R₂ is the phenyl group (compounds 1a, 3a, 4a, 5a and 6a), the        phenylethyl group (compounds 1b, 3b, 4b, 5b and 6b) or the        methyl group (compounds 1c, 3c, 4c, 5c and 6c).

EXAMPLE 1 Preparation of Compound 6a

1) Preparation of Compound 1a

This aminophosphinic acid derivative is prepared according to theprocedure described by Baylis [18], and the enantiomer of Rconfiguration is then obtained via recrystallization according to theprotocol reported by Baylis [18].

2) Preparation of Compound 2a

This compound is obtained according to the procedure published byVillieras et al. [19]. The product obtained was characterized by NMR:

¹H-NMR (250 MHz, CDCl₃): 7.03 (t, 1H), 5.93 (m, 1H), 4.1 (m, 2H), 2.6(m, 1H), 2.34 (m, 2H), 1.95 (s, 3H), 1.82 (m, 1H), 1.18 (t, 3H).

3) Preparation of Compound 3a

A mixture of compound 1a (3.2 g, 10 mmol) and of hexamethyldisilazane(10.5 mL, 50 mmol) under a flow of argon is heated at 110° C. for 3hours. Compound 2 (5.5 g, 12 mmol) is added at this temperature and thissolution is stirred for 4 hours at 90° C. This solution is cooled to 70°C., 10 mL of absolute ethanol EtOH are added dropwise thereto and themixture is stirred at 70° C. for 30 minutes. After evaporating off thesolvents, the residue is dissolved in 5% NaHCO₃ (10 mL) and 5 mL ofhexane. After 3 extractions with ethyl acetate EtOAc (3×5 mL), the crudeproduct is obtained after evaporating off the solvent. Purification on acolumn of silica, using a chloroform/methanol/acetic acid mixture(7/0.3/0.3) as mobile phase, gives 4 g of pure compound 3a, in the formof a white solid (89% yield).

The NMR characterization of this product is based on COSY, TOCSY andHMQC experiments:

¹H-NMR (250 MHz, CDCl₃): 1.27 (t, ³J_(HH)=7.1 Hz, 3H, CH₂CH₃), 1.95-3.00(m, 5H, PCH(CH₂)₂, PhCHH), 3.13-3.45 (m, 2H, PCH, PhCHH), 4.02-4.31 (m,2H, CH₂CH₃), 4.34-4.63 (m, 1H, PCH), 4.78-5.05 (m, 2H, OCH₂Ph), 5.71 (d,1H, NH, ³J_(HH)=11.3 Hz, I), 5.78 (d, 1H, NH, ³J_(HH)=10.8 Hz, II),6.91-6.99 (d, 1H, C═CH), I/II), 7.02-7.34 (m, 10H, aryl).

¹³C-NMR (62 MHz, CDCl₃): 14.2/14.2 (CH₂—CH₃), 26.2/26.5 (PCHCH₂),32.7/32.8 (PCHCH₂CH₂), 34.5 (CH₂Ph), 41.8 (d, ¹J_(PC)=87.7 Hz, PCHC, I),43.1 (d, ¹J_(PC)=87.3 Hz, PCHC, II), 50.5 (d, ¹J_(PC)=99.7 Hz, PCHN, I),50.5 (d, ^(I)J_(PC)=100.5 Hz, PCHN, II), 61.1/61.2 (CH₂CH₃), 66.8(OCH₂Ph), 126.6, 127.7, 127.8, 127.9, 127.9, 128.4, 128.4, 129.4, 132.2,132.3, 132.9, 136.5, 136.6, 137.1, 137.3, (aryls), 148.1 (d, ²J_(PC)=8.7Hz, ═CCO, I), 148.1 (d, ²J_(PC)=9 Hz, ═CCO, II), 156.1 (d, ²J_(PC)=5.5Hz, CONH, I), 156.2 ((d, ²J_(PC)=5.7 Hz, CONH, II), 165.3 (d,²J_(PC)=2.7 Hz, COOEt).

³¹P-NMR (100 MHz, CDCl₃): 46.89, 48.13.

The indications I and II correspond to the different diastereoisomers.

Elemental Analysis:

Theoretical values:

C: 61.80%, H: 6.27%, N: 3.00%.

Experimental values:

C: 61.89%, H: 6.23%, N: 2.98%

4) Preparation of Compound 4a

Compound 3a (1.4 g, 3.06 mmol) and NiCl₂.6H₂O (1.09 g, 9.2 mmol) aredissolved in a mixture of THF (12.4 mL)/methanol (7.7 mL). NaBH₄ (0.58g, 15.4 mmol) is added portionwise to this solution over 30 minutes, at−30° C. This mixture is stirred for a further 10 minutes at −30° C. Thesolvents are evaporated off and the product is extracted into a mixtureof EtOAc (25 mL) and 1N HCl (20 mL, pH 1).

The organic phase is collected and washed with water (10 mL) and thendried with Na₂SO₄. After evaporating off the solvents, the product ispurified on a column of silica with a chloroform/methanol/acetic acid(7/0.3/0.3) mobile phase. 1.28 g of compound 4a are obtained (91%yield).

The analysis by negative-mode mass spectrometry (mass observedMH⁻=458.48, expected mass=459.47) is in accordance with the chemicalstructure of compound 4a.

Elemental Analysis:

Theoretical values:

C: 61.78%, H: 6.63%, N: 3.00%

Experimental values:

C: 61.98%, H: 6.31%, N: 3.08%

5) Preparation of Compound 5a

The adamantylation of compound 4a is performed according to the protocoldescribed by Yiotakis et al. [14].

1-Adamantyl bromide (538 mg, 2.5 mmol) and Ag₂O (577 mg), divided into 5equal portions, are added over 1 hour to a solution of compound 4a (1.03g, 2.24 mmol) in chloroform. After 2 hours, 0.5 eq of AdBr and 0.5 eq ofAg₂O are added and the mixture is refluxed for 10 hours. Afterevaporating off the solvents, the crude product is purified on a columnof silica, using a chloroform/isopropanol mixture (9.8/0.2) as mobilephase. Compound 5a is obtained in pure form in a yield of 96% (1.27 g).

Analysis by mass spectrometry, positive mode: mass observed MH⁺=594.21,expected mass=593.1.

Elemental Analysis:

Theoretical values:

C: 67.76%, H: 7.53%, N: 2.32%

Experimental values:

C: 67.49%, H: 7.58%, N: 2.24%

6) Preparation of Compound 6a

After diluting compound 5a (1.1 g, 1.85 mmol) in methanol (20 mL), 2 mLof 4N NaOH are added. After stirring for 6 hours, TLC monitoring of thereaction confirms the complete saponification of the starting material.After evaporating off the solvent, the product is taken up in a mixtureof water (10 mL) followed by addition of EtOAc (15 mL) and acidificationto pH 1 with 1N HCl. The residue is taken up into the organic phase andthe extraction procedure is repeated twice. The combined organic phasesare dried over Na₂SO₄ and the solvents are then evaporated off. The purecompound 6a is obtained in a yield of 94% (0.98 g).

Analysis by mass spectrometry, positive mode: mass observed MH⁺=566.15,expected mass=565.26.

Elemental Analysis:

Theoretical values:

C: 67.95%, H: 7.13%, N: 2.48%

Experimental values:

C: 67.64%, H: 7.30%, N: 2.40%

EXAMPLE 2 Preparation of Compound 6b

The same procedure as in Example 1 is followed to prepare compound 6b,starting with compound 1b, which is prepared according to the proceduredescribed in [18].

The analysis by mass spectrometry of compound 6b gave the followingresults: expected mass=579.27, mass observed MH⁺=580.29.

EXAMPLE 3 Preparation of Compound 6c

The same procedure as in Example 1 is followed to prepare compound 6c,starting with compound 1c, which is prepared according to the proceduredescribed in [18].

Analysis by mass spectrometry of compound 6c gave the following results:expected mass=489.23, observed mass MH⁺=490.11.

EXAMPLE 4 Preparation of the Pseudopeptide G of Formula

This pseudopeptide was synthesized on a solid phase using a standardprotocol of solid-phase peptide synthesis. Wang resin substituted withan Fmoc-Trp (732 mg, 0.58 mmol) is suspended in N-methylpyrrolidone NMP(5 mL) and stirred for 5 minutes. After removal of the NMP byfiltration, 10 mL of piperidine at a concentration of 20% in NMP areadded and the mixture is stirred for 15 minutes. After filtration, theresin is washed with the following solvents: NMP (7×10 mL), CH₂Cl₂ (3×10mL) and Et₂O (2×10 mL). 2 ml of NMP, diisopropylethylamine DIEA (749 mg,5.76 mmol) and compound 6a (360 mg, 0.64 mmol) diluted in NMP (2 mL) and2-(1H)benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphateHBTU (730 mg, 1.92 mmol, diluted in 3 mL of NMP) are then added to thereactor. The mixture is stirred for 24 hours. After filtration, theresin is washed with NMP (4×7 mL) and CH₂Cl₂ (5×7 mL). A trifluoroaceticacid TFA/CH₂Cl₂/H₂O/triisopropylsilane (90/7.5/1.25/1.25) solution isthen added to the reactor and the mixture is stirred for 3 hours(deprotection phase). After filtration, the filtrate containingpseudopeptide G is recovered, the solvent is evaporated off and theproduct is dissolved in H₂O. After freeze-drying, pseudopeptide G ispurified by reverse-phase HPLC (Vydac column, C18, semi-preparative).

FIG. 2 shows the chromatogram obtained. In this figure, 4 peakscorresponding to the 4 diastereoisomers present in pseudopeptide G areobserved (these 4 peaks have an identical mass spectrum, mass observedMH⁺=618.23, expected mass=617.23). Only peak 1 shows inhibitory powerwith respect to ACE.

The NMR characterization of pseudopeptide G, peak 1 HPLC, is based onCOSY, TOCSY and HMQC experiments:

¹H-NMR (250 MHz, DMSO) 4.93 (d, 2H, CH₂—O-Ph), 4.01 (m, 1H, CH—CH₂-Ph),2.77 (m, 1H, CH—CH₂-Ph), 3.08 (m, 1H, CH—CH₂-Ph), 3.09(α-pseudo-Proline), 1.77 (β-pseudo-Proline), 1.56 (γ-pseudo-Proline),1.79 (δ-pseudo-Proline), 2.49 (ε-pseudo-Proline), 4.50 (α-Trp), 3.13(β,β-Trp), 7.6 NH, 8.3 NH, aryls 7.35-6.9.

EXAMPLES 5 to 7 Preparation of Pseudopeptides B, C and D of Formulae

Pseudopeptides B, C and D were synthesized on a solid phase with Wangresins substituted with alanine (B), proline (C) and arginine (D), usingthe protocol described for the preparation of pseudopeptide G. HPLCpurification of these pseudopeptides made it possible to isolatediastereoisomers of these pseudopeptides capable of inhibiting ACE.

Analysis of the pseudopeptides by mass spectrometry confirms thestructure of these pseudopeptides.

Pseudopeptide B: expected mass 502.19; observed mass 503.21.

Pseudopeptide C: expected mass 528.53; observed mass 529.11.

Pseudopeptide D: expected mass 587.25; observed mass 587.24.

EXAMPLES 8 and 9 Preparation of Pseudopeptides E and F of Formulae

Pseudopeptides E and F are obtained via solid-phase synthesis, startingwith compounds 6b and 6c and following the protocol described for thepreparation of pseudopeptide G. After purification by reverse-phase C18HPLC, the first fraction collected for each of these pseudopeptides wasfound to be capable of inhibiting ACE.

Analysis by mass spectrometry gave the following results:

Pseudopeptide E: expected mass 541.20; observed mass 542.26.

Pseudopeptide F: expected mass 631.24; observed mass 632.26.

EXAMPLE 10 Determination of the Inhibition Constants of Pseudopeptides Ato G with Respect to the N-Terminal and C-Terminal Sites of ACE

Human recombinant ACE is used for this determination. Curves ofinhibition of ACE with pseudopeptides A to G are obtained using thequenched-fluorescence substrate Mca-Ala: Mca-Ala-Ser-Asp-Lys-DpaOH (Mca:7-methoxy-coumarin-2-acetic acid; DpaOH:N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl).

The first fraction collected during the purification of each of thepseudopeptides A to G by HPLC (peak 1 of FIG. 2 for pseudopeptide G) isused for these tests.

From the inhibition profiles obtained with this substrate, for eachpseudopeptide A to G, it is possible to determine the constants Ki N andKi C by following the procedure described by Dive et al. [8].

The inhibition experiments were performed at 25° C., pH 6.8, 50 mMHEPES, 10 mM, CaCl₂, 200 mM NaCl.

The results obtained are given in the attached Table 1.

The same test with pseudopeptide A not comprising the pseudoprolineresidue was performed for comparative purposes. The results obtained arealso given in Table 1. This pseudopeptide was prepared from thephosphinic block ZPhe[PO(OAd)—CH₂]AlaOH, described by Yiotakis et al.[14], followed by coupling of this block to a Wang resin substitutedwith the residue Fmoc-Ala.

Study of the inhibitory effects of pseudopeptides A to G on ACE andcomparison of their affinity towards the N-terminal site (Ki N) and theC-terminal site (Ki C) of ACE (Table 1) allow the following conclusionsto be drawn.

1°) Pseudopeptides A and B: As regards the affinity for the two activesites of ACE, the presence of a pseudoproline residue appears to be muchless favourable than that of a pseudoalanine residue. On the other hand,the presence of a pseudoproline residue in position P1′ of thesephosphinic pseudopeptides gives access to selective inhibitors of theC-terminal site of ACE.

This result demonstrates the essential role of the pseudoproline residueto control the selectivity of the inhibitors with respect to theC-terminal site of ACE.

2°) Pseudopeptides B, C, D and G: The modifications of position P2′ withthe residues alanine, proline, arginine and tryptophan demonstrate thatthe nature of the side chain in this position is also an essentialfactor for selectivity. The presence of the proline residue(pseudopeptide C) generates a powerful but sparingly selective inhibitorof the N and C sites of ACE. On the other hand, the presence of atryptophan residue makes it possible to obtain an extremely selectiveinhibitor (pseudopeptide G) of the C-terminal site of ACE.

3°) Pseudopeptides E and F: The substitution of a pseudophenylalanineresidue in position P1 of the pseudopeptides with a pseudoalanine orpseudo-homo-phenylalanine residue leads to pseudopeptides that are lesspowerful and less selective than pseudopeptide G. This last resultdemonstrates a lesser importance of the position P1 of the inhibitorswith respect to the selectivity.

The study of the pseudopeptides A to G makes it possible to concludethat, in the pseudopeptides of the invention, each position P1, P1′ andP2′ contributes towards the selectivity of the interactions. Thepresence in pseudopeptide G of the pseudophenylalanine, pseudoprolineand tryptophan residues gives it particularly pronounced selectivity.

EXAMPLE 11 Demonstration of the in Vivo Properties of Pseudopeptide G

A study is performed in vivo in order to check the capacity ofpseudopeptide G to inhibit the conversion of angiotensin I intoangiotensin II, and to assess its effects on the cleavage of bradykinin,on the one hand, and of the peptide Ac-SDKP, on the other hand.

1) Study Protocol

This study is performed on batches of male C57BL6/J mice (Iffa Credo)weighing 20 to 23 g each, each batch comprising 6 mice.

To do this, the mice are anaesthetized by means of an intraperitonealadministration of sodium pentobarbital (Sanofi) at a dose of 80 mg/kg ofbody weight. The right carotid artery is isolated and a catheter (PE10,0.28×0.61, A-M Systems, Inc.) is inserted into this artery to allow thewithdrawal of blood samples, while another catheter (FEP, 0.12×0.67,Carnegie Medecin) is inserted into the ipsilateral jugular vein to allowthe administration of the substances. Throughout the experiment, thebody temperature of the mice is maintained at 38° C.

In a first stage, the following are administered to the mice of the samebatch, via perfusion over 30 minutes:

-   -   either 50 μL of an isotonic solution (adjusted to pH 7        containing an amount of pseudopeptide G corresponding to a dose        of 0.9, 3, 10 or 30 mg/kg of body weight,    -   or 50 μL of physiological saline,    -   or alternatively 50 μL of a solution containing an amount of        perindopril corresponding to a dose of 10 mg/kg of body weight,        this substance being a powerful mixed inhibitor of ACE        (Servier).

Next, the following is administered as a bolus:

-   -   either a mixture comprising 2 μg of unlabelled angiotensin I and        21 μCi of ³H-angiotensin I in 50 μL of isotonic solution,    -   or a mixture comprising 2 μg of unlabelled Ac-SDKP and 17 μCi of        ³H-Ac-SDKP in 50 μL of isotonic solution,    -   or a mixture comprising 2 μg of unlabelled bradykinin and 11 μCi        of ³H-bradykinin in 50 μL of isotonic solution.

Samples of arterial blood, of about 50 μl, are withdrawn 30, 60 and 90seconds after injection of the angiotensin I mixture or of thebradykinin mixture to the mice that received these mixtures, and 1, 5,10 and 15 minutes after the start of injection of the Ac-SDKP mixture tothe mice that received the latter mixture. In all cases, the blood iscollected in preweighed polypropylene tubes containing 40 μL of water,10 μL of 80% TFA and 1 μL of heparin. The exact amounts of bloodwithdrawn are determined by reweighing the tubes. After addition of 195μL of distilled water, these tubes are placed in an ice bath for 10minutes and the samples are centrifuged at 4° C. to obtain plasmaextracts.

The analysis of these extracts is performed by liquid chromatographyusing an HPLC system (Perkin Elmer 200) linked to a radioelementdetector (Z 500-4 cell, Berthold). The chromatographic separations areperformed on a Kromasil C18 column (AIT), by injection of 50 μL ofsamples and using the following mobile phases and elution gradients:

Angiotensin I Analysis:

Mobile Phase:

-   -   solvent A: CH₃CN/H₂O/TFA (10/90/0.1)    -   solvent B: CH₃CN/H₂O/TFA (90/10/0.1)

Elution Gradients:

-   -   0-30 min: 0-30% B    -   30-35 min: 30-100% B

Bradykinin Analysis:

Mobile Phase:

-   -   solvent A: CH₃CN/H₂O/TFA (10/90/0.1)    -   solvent B: CH₃CN/H₂O/TFA (90/10/0.1)

Elution Gradients:

-   -   0-30 min: 0-25% B    -   30-35 min: 25-100% B

Ac-SDKP Analysis:

Mobile Phase:

-   -   solvent A: H₂O/TFA (100/0.1)    -   solvent B: CH₃CN/H₂O/TFA (90/10/0.1)

Elution Gradients:

-   -   0-30 min: 0-30% B    -   30-35 min: 30-100% B

The peaks eluted are identified by comparison of their retention timeswith those shown by the unlabelled substrates (angiotensin I, bradykininand Ac-SDKP) and by the expected cleavage products (angiotensin II, BK(1-7) and BK (1-5)).

The assays are performed by integration of the area under thecorresponding peaks of the chromatogram. The values thus obtained arenormalized as a function of the weight of blood taken from each mouse.

The statistical comparisons of the results are performed via thenon-parametric Mann-Whitney U test (Statview 5 software).

2) Results

FIG. 3 shows, in the form of a bar chart, the mean±SD values of theangiotensin II/angiotensin I ratio obtained in the case of the mice thatreceived 0.9, 3, 10 and 30 mg of pseudopeptide G per kg of body weight(bars 0.9, 3, 10 and 30, respectively) and also the mean±SD valueobtained for this same ratio in the control mice (bar T), i.e. the micethat received 50 μl of physiological saline. In this figure, *corresponds to p<0.05, while ** corresponds to p<0.01, by comparison tothe control mice.

FIG. 3 demonstrates the fact that pseudopeptide G is capable ofinhibiting in vivo the cleavage of angiotensin I to angiotensin II andthat this inhibition is dose-dependent. Thus, the angiotensinII/angiotensin I ratio measured in the control mice is reduced by 50% inthe case of the mice treated with 0.9 mg of pseudopeptide G per kg ofbody weight, and by 90% in the case of the mice treated with 30 mg ofpseudopeptide G per kg of body weight.

FIG. 4 illustrates, in the form of a bar chart, the mean±SD percentagesof protection of bradykinin obtained in the case of the mice thatreceived 10 mg of pseudopeptide G per kg of body weight (bar G) and inthe case of the control mice (bar T), i.e., in this case, the mice thatreceived 10 mg of perindopril per kg of body weight. In this figure, **corresponds to p<0.01 by comparison to the control mice.

As may be seen in FIG. 4, with the average degree of protection ofbradykinin with perindopril arbitrarily set at 100%, this degree ofprotection is only 9.2% in the case of the mice which received 10 mg ofpseudopeptide G per kg of body weight. This pseudopeptide thus appearsto afford only a very moderate prevention of cleavage of bradykinin invivo.

FIG. 5 shows, also in the form of a bar chart, the mean±SD bloodconcentrations (expressed in pmol/g of blood) of labelled exogenousAc-SDKP peptide obtained in the case of the mice that received 10 mg ofpseudopeptide G per kg of body weight (bar G) and in the case of thecontrol mice (bar T), i.e. the mice that received 50 μl of physiologicalsaline. In this figure, ** corresponds to p<0.01 by comparison to thecontrol mice.

FIG. 5 also shows, for comparative purposes, the mean±SD bloodconcentration of labelled exogenous Ac-SDKP peptide in the case of themice treated with 10 mg of the pseudopeptide RXP407 (described as beinga selective inhibitor of the N-terminal site of ACE in references [7]and [8]) per kg of body weight (bar R) and following an identicaloperating protocol.

As shown by FIG. 5, pseudopeptide G, at a dose of 10 mg/kg of bodyweight, appears to have no significant effect on the cleavage of thepeptide Ac-SDKP, whereas RXP407 increases the plasmatic content of thispeptide 16-fold relative to that observed in the case of the controlmice.

Thus, in vivo, pseudopeptide G very efficiently inhibits the conversionof angiotensin I to angiotensin II, without veritably preventing thedegradation of bradykinin, and-even less so that of the peptide Ac-SDKP.TABLE 1 Pseudo- Ki N Ki C Formulae peptide nM (10⁻⁹M) nM (10⁻⁹M)

A 0.8 0.8

B 450 20

C 60 4

D 200 9

E 8000 60

F 8000 60

G 10000 3

References

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[2] Linz W., Wiemer G., Gohlke P., Unger T., and Scholkens B. A., 1995,Pharmacol. Rev. 47(1), 25-49.

[3] Soubrier F., Alhenc-Gelas F., Hubert C., Allegrini J., John M.,Tregear G., and Corvol P., 1988, Proc. Natl. Acad. Sci. USA 85(24),9386-90.

[4] Wei L., Alhenc-Gelas F., Corvol P., and Clauser E., 1991, J. Biol.Chem. 266(14), 9002-8.

[5] Jaspard E., Wei L., and Alhenc-Gelas F. (1993) J. Biol. Chem.268(13), 9496-503.

[6] Azizi M., Rousseau A., Ezan E., Guyene T. T., Michelet S., GrognetJ. M., Lenfant M., Corvol P., and Menard J., 1996, J. Clin. Invest.97(3), 839-44.

[7] WO-A-00/01706.

[8] Dive V., Cotton J., Yiotakis A., Michaud A., Vassiliou S., JiracekJ., Vazeux G., Chauvet M. T., Cuniasse P., and Corvol P. (1991) Proc.Natl. Acad. Sci. USA 96(8), 4330-5.

[9] Junot C., Gonzales M. F., Ezan E., Cotton J., Vazeux G., Michaud A.,Azizi M., Vassiliou S., Yiotakis A., Corvol P., and Dive V., 2001, J.Pharmacol. Exp. Ther. 297(2), 606-11.

[10] FR-A-2 676 059.

[11] EP-A-0 725 075.

[12] Jiracek J., Yiotakis A., Vincent B., Lecoq A., Nicolaou A., CheclerF., and Dive V., Development of highly potent and selective phosphinicpeptide inhibitors of zinc endopeptidase 24-15 using combinatorialchemistry., 1995, J. Biol. Chem. 270(37): 21701-6.

[13] Jiracek J., Yiotakis A., Vincent B., Checler F. and Dive V.,Development of the first potent and selective inhibitor of the zincendopeptidase neurolysin using a systematic approach based oncombinatorial chemistry of phosphinic peptides, 1996, J. Biol. Chem.271(32): 19606-11.

[14] Yiotakis A., Vassiliou S., Jiracek J., and Dive V., Protection ofthe hydroxy-phosphinyl function of phosphinic dipeptides by adamantyl.Application to the solid-phase synthesis of phosphinic peptides, 1996,J. Org. Chem. 61: 6601-6605.

[15] Vassiliou S., Mucha A., Cuniasse P., Georgiadis D., Lucet-LevannierK., Beau F., Kannan R., Murphy G., Knauper V., Rio MC., Basset P.,Yiotakis A., and Dive V., Phosphinic pseudo-tripeptides as potentinhibitors of matrix metalloproteinases: a structure-activity study,1999, J. Med. Chem. 42(14): 2610-20.

[16] Georgiadis D., Vazeux G., Llorens-Cortes C., Yiotakis A., and DiveV., Potent and selective inhibition of zinc aminopeptidase A (EC3.4.11.7, APA) by glutamyl aminophosphinic peptides: importance ofglutamyl aminophosphinic residue in the P1 position, 2000, Biochemistry39(5): 1152-5.

[17] Protective groups in Organic Synthesis, Second Edition, T. W. Greenand P. G. M. Wuts, John Wiley & Sons, Inc., 309-315.

[18] Baylis E. K., Campbell C. D., and Dingwall J. G., 1984, J. Chem.Soc. Perkin. Trans. I, 2845.

[19] Villieras J., Rambaud W. H., and Graff M., 1986, Synth. Commun. 16,149.

[20] Chen H., Noble F., Roques P., and Fournie-Zaluski M. C., 2001, J.Med. Chem. 44, 3523-3530.

1. A method for selectively inhibiting the C-terminal site ofangiotensin I converting enzyme comprising utilizing at least onephosphinic pseudopeptide derivative comprising the amino acid sequenceof formula (I) below:

wherein, R₂ and R₃, which are identical or different, represent the sidechain of a natural or unnatural amino acid, the sequence:

also possibly forming the Pro (proline) residue, and R₅ represents ahydrogen atom, a pharmacologically acceptable counterion, or a groupthat can form an in vivo hydrolysable phosphinic ester;
 2. A method forselectively inhibiting the C-terminal site of angiotensin I convertingenzyme comprising utilizing a phosphinic pseudopeptide derivativecorresponding to formula (II) below:

wherein, R₁ represents a protecting group for an amine function, or anamino acid or a peptide protected with a protecting group for an aminefunction, R₂ and R₃, which may be identical or different, represent theside chain of a natural or unnatural amino acid, the sequence:

also possibly forming the Pro residue, R₄ represents a hydrogen atom ora pharmacologically acceptable counterion, and R₅ represents a hydrogenatom, a pharmacologically acceptable counterion, or a group that canform an in vivo hydrolysable phosphinic ester;
 3. The method of claim 2,wherein R₁ represents a protecting group for an amine function chosenfrom acetyl and benzyloxycarbonyl groups.
 4. The method of claim 1,wherein R₂ represents the benzyl, methyl or phenylethyl group.
 5. Themethod of claim 1, wherein R₃ represents the side chain of alanine,arginine or tryptophan.
 6. The method of claim 1, wherein the sequence—NH—CH(R₃)—CO— forms the Pro residue:


7. The method of claim 1, wherein R₄ and/or R₅ represent(s) a hydrogenatom.
 8. The method of claim 2, wherein the phosphinic pseudopeptidederivative corresponds to the formula:


9. A phosphinic pseudopeptide derivative comprising the amino acidsequence of formula (I) below:

wherein, R₂ represents the side chain of a natural or unnatural aminoacid, the sequence:

forms the Pro residue:

R₄ represents a hydrogen atom or a pharmacologically acceptablecounterion, and R₅ represents a hydrogen atom, a pharmacologicallyacceptable counterion, or a group than can form an in vivo hydrolysablephosphinic ester.
 10. A phosphinic pseudopeptide derivativecorresponding to formula (II) below:

wherein, R₁ represents a protecting group for an amine function, or anamino acid or a peptide protected with a protecting group for an aminefunction, R₂ represents the side chain of a natural or unnatural aminoacid, the sequence:

forms the Pro residue:

R₅ represents a hydrogen atom, a pharmacologically acceptablecounterion, or a group that can form an in vivo hydrolysable phosphinicester.
 11. A phosphinic pseudopeptide derivative of formula:


12. A pharmaceutical composition comprising at least one phosphinicpseudopeptide derivative as claimed in claim
 9. 13. A pharmaceuticalcomposition, in which the phosphinic pseudopeptide derivativecorresponds to the formula:


14. A process for preparing a pseudopeptide of formula:

wherein R₁ represents a protecting group for an amine function, or anamino acid or a peptide protected with a protecting group for an aminefunction, R₂ and R₃, which may be identical or different, represent theside chain of a natural or unnatural amino acid, the sequence:

also possibly forming the Pro residue, and R₄ and R₅ represent ahydrogen atom; which comprises the following steps: 1) reacting acompound of formula (III):

in which R₁ and R₂ are as defined above, with the compound of formula(IV):

in which Ac represents the acetyl group and Et represents the ethylgroup, to obtain the compound of formula (V):

2) converting compound (V) into compound (VI) by reacting compound (V)with sodium borohydride:

3) protecting the hydroxyl group of compound (VI) with a protectinggroup R₅, for example the adamantyl group Ad, to give the compound offormula (VII):

4) saponifying compound (VII) to give the compound of formula (VIII):

5) coupling the compound of formula (VIII) with the amino acid offormula (IX) or (X):

in which R₃ is as defined above, and 6) removing the protecting groupAd.
 15. A process as claimed in claim 14, wherein the peptide couplingstep 5) is performed via solid-phase peptide synthesis wherein the solidphase is a resin substituted with the amino acid of formula (IX) or (X).16. A process for preparing a pseudopeptide of formula:

wherein, R₁ represents a protecting group for an amine function, or anamino acid or a peptide protected with a protecting group for an aminefunction, R₂ and R₃, which may be identical or different, represent theside chain of a natural or unnatural amino acid, the sequence:

also possibly forming the Pro residue, R₄ represents a hydrogen atom,and R₅ represents a group that can form an in vivo hydrolysablephosphinic ester; wherein the phosphinic function of the pseudopeptideobtained via the process of claim 14 is esterified by coupling with analcohol of formula R₅OH or by reaction with a halide of formula R₅X inwhich X represents a halogen atom.
 17. A compound of formula (VIII):

wherein R₁ represents a protecting group for an amine function or anamino acid or a peptide protected with an amine function, and R₂represents the side chain of a natural or unnatural amino acid.
 18. Themethod of claim 2, wherein R₂ represents the benzyl, methyl orphenylethyl group.
 19. The method of claim 2, wherein R₃ represents theside chain of alanine, arginine or tryptophan.
 20. The method of claim2, wherein the sequence —NH—CH(R₃)—CO— forms the Pro residue:


21. The method of claim 2, wherein R₄ and/or R₅ represent(s) a hydrogenatom.
 22. A pharmaceutical composition comprising at least onephosphinic pseudopeptide derivative as claimed in claim
 10. 23. Apharmaceutical composition comprising at least one phosphinicpseudopeptide derivative as claimed in claim
 11. 24. A process forpreparing a pseudopeptide of formula:

wherein, R₁ represents a protecting group for an amine function, or anamino acid or a peptide protected with a protecting group for an aminefunction, R₂ and R₃, which may be identical or different, represent theside chain of a natural or unnatural amino acid, the sequence:

also possibly forming the Pro residue, R₄ represents a hydrogen atom,and R₅ represents a group that can form an in vivo hydrolysablephosphinic ester; wherein the phosphinic function of the pseudopeptideobtained via the process of claim 15 is esterified by coupling with analcohol of formula R₅OH or by reaction with a halide of formula R₅X inwhich X represents a halogen atom.